Studies on cartilage formation. XXII. Investigations of certain oxidative metabolic processes in regenerating articular cartilage

The distal articular surface of the femur was surgically removed in 57 dogs. Succinate dehydrogenase and cytochrome oxidase activities were assayed on postoperative days 7, 20, 26, 33 and 70 in the regenerating, chondrifying articular surface and in the granulation tissue adhering to the capsule. In the 70-day samples, the cyanide-induced inhibition of oxygen consumption was determined and enzyme histochemical reactions (cytochrome oxidase, monoamine oxidase, xanthine oxidase, peroxidase and "catalase") were performed. The succinate dehydrogenase activity was the highest in the early postoperative stage in both tissues. This was followed by a definite decrease and a subsequent significant increase in activity when chondrification took place. Measurement of cytochrome oxidase activity could not reveal any convincing result, presumably because of the properties of the tissues studied. The oxygen consumption by the chondrifying articular surface at 70 days was inhibited to about 50% by cyanide, and about 90% inhibition was observed in the tissue adhering to the capsule. The cells of the regenerating articular surface possess cytochrome oxidase and a cyanide- (and sodium azide-) resistant oxidase activity. The enzyme activity of the cartilaginous islets exceeded that of their connective tissue environment. The cytochrome oxidase activity increased in the cells during cartilage differentiation. Presumably, some further cyanide-sensitive and cyanide-resistant oxidases are present in chondroblasts and young chondrocytes.     

Studies on cartilage formation. XVI. Chemical and histochemical assay of lipids in the regenerating articular cartilage.

The articular surface of the distal part of the femur was removed operatively in dogs, and the regenerating articular surface and the GTC were investigated at different stages from the 7th to the 70th postoperative days. During this period cartilage islets arose in the GTAS, while the GTC transformed to connective tissue. At 7 days the lipid content of the tissue was markedly higher than at the other stages studied. Lipids, predominantly triglycerides, were present in extracellular form as well. From the 20th to the 70th day the PL fraction became predominant and, in addition to the pre-existing lecithin, relatively large quantities of lysolecithin, sphingomyelin, phosphatidyl-ethanolamine, phosphatidyl-serine and phosphatidyl-inositol could be gradually demonstrated. Differences were noted in the time of appearance and binding of PLs between the two types of granulation tissue. As time proceeded, the proportion of saturated fatty acids decreased in favour of unsaturated ones. At 70 days, the GTAS contained fatty acids up to C18. About 50% of the fatty acids consisted of C16:1, C18:2 and C18:1. At the same stage, in the GTC C16:1, C18:1 and C20:1 were present in larger amounts. Of the free fatty acids C16:1, C16 and C18 were in predominance in the GTAS and the proportion of fatty acids having more then one double bonds increased with time. In the GTC C16 and C18:1 were in great majority. According to histochemical evidence, the tissues did not contain extracellular lipids from the 20th postoperative day. In the cells, the presence of glycerides, PLs, lipoproteins and cholesterol was demonstrated. In addition, in cartilage precursors of more advanced maturity, a considerable fatty acid positivity was noted.     

The biology of cartilage. II. Invertebrate cartilages: squid head cartilage

In both light and electron microscopes, head cartilage from the squid Loligo pealii strongly resembles vertebrate hyaline cartilage. The tissue is characterized by the presence of irregularly-shaped cells suspended in an abundant matrix. Cell and matrix contents stain metachromatically with cationic dyes such as toluidin blue. Each cell gives off extensions which ramify via a network of channels throughout the matrix. Thereby, a system of inter-connecting canaliculi is established, with many similarities to the intercanalicular systems seen in vertebrate bone and cartilage tissues. In the electron microscope, the squid cartilage cells are seen to have very abundant endoplasmic reticulum and Golgi complex material. Mitochondrial transformations involving loss of cristae, the appearance of filaments in the mitochondrial matrix, and figures suggesting budding, also occur. Nuclear pores are numerous and easily detected. The matrix is characterized by the presence of a system of decussating fibrils which form polygonal figures, with granules usually evident at the points of intersection of fibrils. By chemical analysis the tissue contains 3- and 4-hydroxyproline and hydroxylysine. Preliminary wide single x-ray diffractions show a pattern characteristic for unoriented collagens, with 12 Å (intermolecular) and 2.86 Å (helix) reflections.     

The biology of cartilage. I. Invertebrate cartilages: Limulus gill cartilage

The endoskeletal structure supporting the gill-books of Limulus polyphemus has been investigated by means of light and electron microscopy, chemical analysis and x-ray diffraction. This tissue is a cartilage which has significant correspondences with both vertebrate cartilage and plant tissues. Morphologically, the Limulus cartilage resembles certain cellular vertebrate cartilages with relatively scant matrix, and also certain plant parenchyme, collenchyme and sclerenchyme tissues. Of particular interest, was the observation that during cytoplasmic division, a phragmasome-like structure appears between the daughter cells of the dividing gill cartilage cells. This phragmasome-like structure appears to be a precursor of new matrix (cell-wall) formation between the young chondrocytes, in much the same fashion as its counterpart in plant tissues. Perichondrial cells and underlying chondrocytes contain lipid droplets, abundant glycogen and ribosomes, as do corresponding vertebrate cartilage cells. In some of the Limulus cells, glycogen and ribosomes appear to be admixed with lipid, forming aggregates in which all three materials are in intimate intraparticulate relationship. During molting, the number of ribosomes seen in chondrocytes increases. The tissue contains both hydroxyproline and hydroxylysine, and gives a weak x-ray diffraction pattern.     

Effect of cartilage proteoglycans on human collagenase activities.

No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w, had little effect on collagen degradation as judged by the reconstituted [4C]collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synoviam, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.     

Lack of effect of lysozymes on cartilage proteoglycans.

Certain similarities between lysozyme and testicular hyaluronidase suggested that the putative action of the former in disaggregation of cartilage proteoglycans might be explained by a selective effect of the enzyme on the hyaluronic acid portion of the aggregate. Human lysozyme did not reduce the viscosity of either hyaluronate or of aggregated proteoglycans, it did not reduce the sedimentation velocity of hyaluronate, and it did not alter the chromatographic profile of the aggregate in a system sensitive to the difference between aggregate and its subunit. The role of human cartilage lysozyme in disaggregation of cartilage proteoglycans remains uncertain.     

Studies on cathepsin B in human articular cartilage.

The thiol proteinase cathepsin B (EC 3.4.22.1), previously called cathepsin B1, was assayed in human articular cartilage by its hydrolysis of the synthetic substrate alpha-N-benzoyl-DL-arginine 2-naphthylamide. The enzyme was activated by cysteine and EDTA and completely inhibited by iodoacetamide and HgCl2. It was also partially inhibited by whole human serum. Human osteoarthrotic cartilage had increased activity when compared with normal cartilage. Cathepsin B activity of normal cartilage was age-related, being high in juveniles and declining to low values in adult and elderly individuals. Cathepsin D and cathepsin B both exhibited a zonal variation through the cartilage depth; the surface cells appeared to contain more activity than those close to the subchondral bone.     

Action of rheumatoid synovial collagenase on cartilage collagen. Different susceptibilities of cartilage and tendon collagen to collagenase attack.

The action of purified rheumatoid synovial collagenase on purified cartilage collagen, alpha-1(II)-3, in solution at 25 degrees C has been characterised. The enzyme attacked cartilage collagen in solution producing a 58% reduction in specific viscosity and resulting in the appearance of two reaction products which represented approximately three-quarter and one-quarter fragments of the intact molecule as shown by disc electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. The alpha-chain fragments which comprised each of these components corresponded to molecular weights of approximately 74000 and 21000. Electron microscopy of segment-long-spacing crystallites of the reaction products revealed three-quarter (TC-a) and one-quarter (TC-b) length fragments, and permitted accurate localization of the cleavage locus between bands 41 and 42 (I-41). This cleavage site and the formation of TC-a and TC-b reaction products are very similar to those found for type-I collagen substrates. Cartilage collagen in solution was found to be more resistant to collagenase attack than tendon collagen, the rate of cartilage collagen degradation being six times slower than that for tendon collagen, as judged by viscometry. The mid-point melting temperatures (T-m) for lathyritic cartilage and tendon collagen were 40.5 and 41.5 degrees C, and for the collagenase-produced reaction products 38.5 and 37.5 degrees C, respectively. The significance of these findings is discussed in relation to the structure of type I and II collagens.     

Studies on the polydispersity and heterogeneity of cartilage proteoglycans. Identification of 3 proteoglycan structures in bovine nasal cartilage.

1. Three chondroitin sulphate components were isolated from adult bovine nasal cartilage after treatment with alkaline NaB3H. Average molecular weights of 13000, 18 600 and 28 000 were obtained for chondroitin sulphate species representing 10, 52 and 38% (w/w) of the total chondroitin sulphate respectively. Each chondroitin sulphate pool has a narrow molecular-weight distribution. 2. A proteoglycan subunit preparation, isolated from one nasal cartilage by extraction and density-gradient fractionation in dissociative solvents, partitioned on a CSCl density gradient according to size and composition. Variation of proteoglycan molecular weight across the gradient was directly related to the average chondrotin sulphate chain length, which in turn reflected the relative proportion of the three chondroitin sulphate pools in each proteoglycan fraction. Consideration of proteoglycan molecular parameters, compositions and behaviour on sedimentation leads to a proposal that nasal cartilage contains 3 distinct proteoglycan pools, each of which has a constant number of chondroitin sulphate side chains of different average molecular weight. 3. Molecular-weight distribution parameters for these proteoglycan preparations indicate that all serine residues on the protein core capable of initiating chondroitin sulphate biosynthesis are occupied and that proteoglycan polydispersity results directly from the polydispersity of the attached chondroitin sulphate component.     

Glycosaminoglycan turn-over in articular cartilage.

Glycosaminoglycan turn-over has been studied both in vivo and in vitro, by using sodium [35S]sulphate as a precursor. The in vivo experiments were performed on rabbits and dogs, taking special care to monitor the 35S radioactivity in the serum throughout the experiment and to measure the radioactivity due to unincorporated inorganic [35S]sulphate in cartilage at the end of each experiment, in addition to that due to incorporated sulphate. The inorganic sulphate content of the serum was also determined as well as the distribution coefficient for the inorganic sulphate ion between cartilage and serum. From this information it was possible to calculate accurately the rate of sulphate uptake by cartilage in vivo and hence the turn-over rate. Experiments were then performed in vitro on cartilage from rabbits and dogs and the in vivo and in vitro results were compared. A very good agreement was obtained between the two sets of results. Studies were then carried out under exactly the same in vitro conditions on human articular cartilage and it was thus possible to obtain a turn-over rate for the latter which one could trust was close to the actual in vivo value. The mean half-lives thus obtained varied from 45 days for the young rabbit to 150 days for the adult dog and 800 days for the human femoral head. In human cartilage there were considerable variations in turn-over rate within a single joint as a function of depth below the surface, and between different joints. Thus, while the mean half-life for the human femoral head is 800 days, that for the femoral condyle is 300 days. Cartilage from osteoarthrosic femoral heads did not appear to differ much with respect to sulphate uptake from the normal specimens although the turn-over rates were somewhat higher.     

Matrix vesicles in aging cartilage.

The calcification process that occurs in aging has been studied with the electron microscope in costal and tracheal cartilage of rats and in human costal cartilage. In these tissues, the early stage of the calcification process is induced and regulated by matrix vesicles in the same way as it occurs in epiphyseal cartilage, bone, and dentine. However, the spreading of inorganic substance from vesicles into the surrounding matrix is frequently impaired in aged cartilage, either because of a too low concentration of calcium ions, or because the structure of the cartilage matrix is not suitable for inorganic substance deposition. This shows that matrix vesicles have a calcium affinity and calcium-binding potentiality greater than that of other components of the cartilage matrix. Most matrix vesicles are produced by "Verd?mmerung der Zellen." This degenerative process of the chondrocytes leads also to the formation of pericellular halos consisting of aggregates of amorphous substance and thin filaments. Part of the material that forms these aggregates seems to be produced by disruption of matrix vesicles. Within this disruptive material, thick collagen fibrils can be formed. Moreover, this material seems capable of inducing calcification. These findings suggest that matrix vesicles, by releasing their content into the matrix, can be involved in some way in collagen formation, and that the released material maintains the calcium affinity and calcium-binding property it has within the vesicles.